PROJECT SUMMARY In response to starvation, bacterial species of the orders Bacillales and Clostridiales differentiate into spores. These stress-resistant cell types are metabolically inactive and can remain dormant for years but rapidly germinate and resume growth upon sensing that proper nutrient conditions have returned. Many of these species are human pathogens, and in their dormant state are highly resistant to antibiotics and can withstand otherwise sterilizing treatments like heat and UV irradiation. To better understand how to prevent pathogenic spore-formers from entering this highly durable state, the molecular mechanisms underlying virtually every step in the sporulation pathway have been characterized. However, the equally important process of germination remains less well understood. A more complete molecular characterization of germination will facilitate the development of treatments that can prevent exit from dormancy or trigger premature germination, leaving cells vulnerable to antibacterial therapies. Most endospore-forming bacteria follow a similar germination program that involves a conserved set of factors. The first step involves environmental monitoring by a large family of putative germinant receptors. In Bacillus subtilis, the prototypical receptor is composed of the products of the gerA operon ? GerAA, GerAB, and GerAC. The GerA proteins are required for spore germination in response to L-alanine. How they function and whether or not they act as a nutrient receptor has not been established. Upon sensing germinants, receptors act by an unknown mechanism to release large stores of the small molecule dipicolinic acid (DPA) from the spore core. Water replaces the DPA resulting in partial spore hydration. A conserved membrane complex is required for DPA release but how it is activated is unknown. Finally, conserved cell wall hydrolases packaged in the spore are activated and degrade a thick layer of specialized peptidoglycan known as the spore cortex. Removal of this protective layer allows further core hydration, the onset of metabolic activity, and the resumption of growth. How these enzymes are activated and their substrate specificities remain poorly understood. This proposal seeks to define the molecular underpinning of all three steps in the germination pathway, working from the last temporal step to the first. The specific aims are: Aim 1: Determine the mechanism that activates the cortex-degrading enzyme CwlJ and define its substrate specificity. Aim 2: Investigate the mechanism of germinant detection and signal transduction.